Chemiluminescent optical fiber immunosensor for detection of autoantibodies to ovarian and breast cancer-associated antigens

We report herein the development of an optical fiber based chemiluminescent immunosensor for detection of the native autoimmune response to GIPC-1, a PDZ containing protein involved in regulation of G-protein signaling. The recombinant protein GIPC-1 was expressed in bacteria, purified, refolded and conjugated to the tip of an optical fiber. A human monoclonal 27.B1 IgM isolated from a breast cancer patient, which targets the GIPC-1 protein, was used for calibration of the immunosensor and was detected down to a concentration of 30 pg/ml. We determined that the fiber-optic immunosensor had a detection limit 50 times lower than chemiluminescent ELISA, and approximately 500 times lower than colorimetric ELISA. In addition, sera from 11 ovarian cancer patients, 22 breast cancer patients and asymptomatic controls were tested for the presence of IgM anti-GIPC-1 autoantibodies in their serum using the two methods. The immunosensor assay detected 54% and 77% GIPC-1 positive sera within ovarian and breast cancer patients, respectively, as compared to chemiluminescent ELISA, which only detected 18% and 27%, respectively. We envision that this immunosensor may serve as a diagnostic tool for screening women for ovarian and breast cancer at an early stage, thus increasing their chance of survival.     

Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastases

Radiofrequency (RF) ablation (RFA) is a minimally invasive treatment for colorectal-cancer liver metastases (CLM) in selected nonsurgical patients. Unlike surgical resection, RFA is not followed by routine pathological examination of the target tumor and the surrounding liver tissue. The aim of this study was the evaluation of apoptotic events after RFA. Specifically, we evaluated YO-PRO-1 (YP1), a green fluorescent DNA marker for cells with compromised plasma membrane, as a potential, early marker of cell death. YP1 was applied on liver tissue adherent on the RF electrode used for CLM ablation, as well as on biopsy samples from the center and the margin of the ablation zone as depicted by dynamic CT immediately after RFA. Normal pig and mouse liver tissues were used for comparison. The same samples were also immunostained for fragmented DNA (TUNEL assay) and for active mitochondria (anti-OxPhos antibody). YP1 was also used simultaneously with propidium iodine (PI) to stain mouse liver and samples from ablated CLM. Following RFA of human CLM, more than 90 % of cells were positive for YP1. In nonablated, dissected pig and mouse liver however, we found similar YP1 signals (93.1 % and 65 %, respectively). In samples of intact mouse liver parenchyma, there was a significantly smaller proportion of YP1 positive cells (22.7 %). YP1 and PI staining was similar for ablated CLM. However in dissected normal mouse liver there was initial YP1 positivity and complete absence of the PI signal and only later there was PI signal. Conclusion: This is the first time that YP1 was applied in liver parenchymal tissue (rather than cell culture). The results suggest that YP1 is a very sensitive marker of early cellular events reflecting an early and widespread plasma membrane injury that allows YP1 penetration into the cells.     

Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn⁻ substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd⁻ substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd⁻ substitutions enhance YKD-KD interactions in vitro, whereas Gcn⁻ substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd⁻ substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD.     

TGF‐beta signalling in the adult neurogenic niche promotes stem cell quiescence as well as generation of new neurons

Members of the transforming growth factor (TGF)‐β family govern a wide range of mechanisms in brain development and in the adult, in particular neuronal/glial differentiation and survival, but also cell cycle regulation and neural stem cell maintenance. This clearly created some discrepancies in the field with some studies favouring neuronal differentiation/survival of progenitors and others favouring cell cycle exit and neural stem cell quiescence/maintenance. Here, we provide a unifying hypothesis claiming that through its regulation of neural progenitor cell (NPC) proliferation, TGF‐β signalling might be responsible for (i) maintaining stem cells in a quiescent stage, and (ii) promoting survival of newly generated neurons and their functional differentiation. Therefore, we performed a detailed histological analysis of TGF‐β1 signalling in the hippocampal neural stem cell niche of a transgenic mouse that was previously generated to express TGF‐β1 under a tetracycline regulatable Ca‐Calmodulin kinase promoter. We also analysed NPC proliferation, quiescence, neuronal survival and differentiation in relation to elevated levels of TGF‐β1 in vitro and in vivo conditions. Finally, we performed a gene expression profiling to identify the targets of TGF‐β1 signalling in adult NPCs. The results demonstrate that TGF‐β1 promotes stem cell quiescence on one side, but also neuronal survival on the other side. Thus, considering the elevated levels of TGF‐β1 in ageing and neurodegenerative diseases, TGF‐β1 signalling presents a molecular target for future interventions in such conditions.     

Advances in Ecological Speciation: an integrative approach

The role of natural selection in promoting reproductive isolation has received substantial renewed interest within the last two decades. As a consequence, the study of ecological speciation has become an extremely productive research area in modern evolutionary biology. Recent innovations in sequencing technologies offer an unprecedented opportunity to study the mechanisms involved in ecological speciation. Genome scans provide significant insights but have some important limitations; efforts are needed to integrate them with other approaches to make full use of the sequencing data deluge. An international conference ‘Advances in Ecological Speciation’ organized by the University of Porto (Portugal) aimed to review current progress in ecological speciation. Using some of the examples presented at the conference, we highlight the benefits of integrating ecological and genomic data and discuss different mechanisms of parallel evolution. Finally, future avenues of research are suggested to advance our knowledge concerning the role of natural selection in the establishment of reproductive isolation during ecological speciation.     

Consensus between Pipelines in Structural Brain Networks

Structural brain networks may be reconstructed from diffusion MRI tractography data and have great potential to further our understanding of the topological organisation of brain structure in health and disease. Network reconstruction is complex and involves a series of processesing methods including anatomical parcellation, registration, fiber orientation estimation and whole-brain fiber tractography. Methodological choices at each stage can affect the anatomical accuracy and graph theoretical properties of the reconstructed networks, meaning applying different combinations in a network reconstruction pipeline may produce substantially different networks. Furthermore, the choice of which connections are considered important is unclear. In this study, we assessed the similarity between structural networks obtained using two independent state-of-the-art reconstruction pipelines. We aimed to quantify network similarity and identify the core connections emerging most robustly in both pipelines. Similarity of network connections was compared between pipelines employing different atlases by merging parcels to a common and equivalent node scale. We found a high agreement between the networks across a range of fiber density thresholds. In addition, we identified a robust core of highly connected regions coinciding with a peak in similarity across network density thresholds, and replicated these results with atlases at different node scales. The binary network properties of these core connections were similar between pipelines but showed some differences in atlases across node scales. This study demonstrates the utility of applying multiple structural network reconstrution pipelines to diffusion data in order to identify the most important connections for further study.     

Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin,and Src

In osteoclasts (OCs) podosomes are organized in a belt, a feature critical for bone resorption. Although microtubules (MTs) promote the formation and stability of the belt, the MT and/or podosome molecules that mediate the interaction of the two systems are not identified. Because the growing “plus” ends of MTs point toward the podosome belt, plus-end tracking proteins (+TIPs) might regulate podosome patterning. Among the +TIPs, EB1 increased as OCs matured and was enriched in the podosome belt, and EB1-positive MTs targeted podosomes. Suppression of MT dynamic instability, displacement of EB1 from MT ends, or EB1 depletion resulted in the loss of the podosome belt. We identified cortactin as an Src-dependent interacting partner of EB1. Cortactin-deficient OCs presented a defective MT targeting to, and patterning of, podosomes and reduced bone resorption. Suppression of MT dynamic instability or EB1 depletion increased cortactin phosphorylation, decreasing its acetylation and affecting its interaction with EB1. Thus, dynamic MTs and podosomes interact to control bone resorption.     

Predicting the sensitivity of butterfly phenology to temperature over the past century

Studies to date have documented substantial variation among species in the degree to which phenology responds to temperature and shifts over time, but we have a limited understanding of the causes of such variation. Here, we use a spatially and temporally extensive data set (ca. 48 000 observations from across Canada) to evaluate the utility of museum collection records in detecting broad‐scale phenology‐temperature relationships and to test for systematic differences in the sensitivity of phenology to temperature (days °C^?1) of Canadian butterfly species according to relevant ecological traits. We showed that the timing of flight season predictably responded to temperature both across space (variation in average temperature from site to site in Canada) and across time (variation from year to year within each individual site). This reveals that collection records, a vastly underexploited resource, can be applied to the quantification of broad‐scale relationships between species' phenology and temperature. The timing of the flight season of earlier fliers and less mobile species was more sensitive to temperature than later fliers and more mobile species, demonstrating that ecological traits can account for some of the interspecific variation in species' phenological sensitivity to temperature. Finally, we found that phenological sensitivity to temperature differed across time and space implying that both dimensions of temperature will be needed to translate species' phenological sensitivity to temperature into accurate predictions of species' future phenological shifts. Given the widespread temperature sensitivity of flight season timing, we can expect long‐term temporal shifts with increased warming [ca. 2.4 days °C^?1 (0.18 SE)] for many if not most butterfly species.     

A Spontaneous Animal Model of Intestinal Dysmotility Evoked by Inflammatory Nitrergic Dysfunction

Background and Aims

Recent reports indicate the presence of low grade inflammation in functional gastrointestinal disorders (FGID), in these cases often called “post-inflammatory” FGIDs. However, suitable animal models to study these disorders are not available. The Biobreeding (BB) rat consists of a diabetes-resistant (BBDR) and a diabetes-prone (BBDP) strain. In the diabetes-prone strain, 40–60% of the animals develop diabetes and concomitant nitrergic dysfunction. Our aim was to investigate the occurrence of intestinal inflammation, nitrergic dysfunction and intestinal dysmotility in non-diabetic animals.

Methods

Jejunal inflammation (MPO assay, Hematoxylin&Eosin staining and inducible nitric oxide synthase (iNOS) mRNA expression), in vitro jejunal motility (video analysis) and myenteric neuronal numbers (immunohistochemistry) were assessed in control, normoglycaemic BBDP and diabetic BBDP rats. To study the impact of iNOS inhibition on these parameters, normoglycaemic BBDP rats were treated with aminoguanidine.

Results

Compared to control, significant polymorphonuclear (PMN) cell infiltration, enhanced MPO activity, increased iNOS mRNA expression and a decreased ratio of nNOS to Hu-C/D positive neurons were observed in both normoglycaemic and diabetic BBDP rats. Aminoguanidine treatment decreased PMN infiltration, iNOS mRNA expression and MPO activity. Moreover, it restored the ratio of nNOS to Hu-C/D positive nerves in the myenteric plexus and decreased the abnormal jejunal elongation and dilation observed in normoglycaemic BBDP rats.

Conclusions

Aminoguanidine treatment counteracts the inflammation-induced nitrergic dysfunction and prevents dysmotility, both of which are independent of hyperglycaemia in BB rats. Nitrergic dysfunction may contribute to the pathophysiology of “low-grade inflammatory” FGIDs. Normoglycaemic BBDP rats may be considered a suitable animal model to study the pathogenesis of FGIDs.